Smad3 mediates TGF-beta induced apoptosis through the control of survivin expression in human breast cancer cells

Transforming growth factor-beta (TGF-beta) pathway initiates signaling by assembling receptor complexes that activate Smad transcription factors. TGF-beta plays a key role in cell proliferation, differentiation and apoptosis. The mechanism of TGF-beta induced apoptosis has not been elucidated. We have approached the blockade of Smad3 by using stable ectopic anti-sense Smad3 (AS-Smad3) expression in a breast cancer cell line to understand how the loss of Smad3 affects on the inhibitory TGF-beta mediated cell death. In AS-Smad3 MCF-7E cells, we found that the expression of Smad3 mRNA and protein was reduced up to ∼40--45% and were less sensitive to autocrine TGF-beta responses than empty vector control using the p3TP-Lux reporter construct. We demonstrated that AS-Smad3 cells showed enhanced anchorage independent growth and cell proliferation. Furthermore, AS-Smad3 cells exhibited delayed apoptosis. We examined whether increased cell survival is involved in the regulation of inhibitor of apoptotic protein (IAP), survivin. We demonstrated that the expression of survivin in AS-Smad3 cells was increased and was reduced by inhibitory TGF-beta treatment and rescued by reintroducing wild type Smad3 plasmid in AS-Smad3 cells. In addition, we exhibited that TGF-beta signaling regulates the 5' flanking region (-124 to -113) of human survivin gene in a Smad3 dependent manner. These results indicate that the role of Smad3 in antiproliferative TGF-beta pathway converges into the down-regulation of survivin expression resulting in acceleration of tumorigenesis and inhibitory TGF-beta induced apoptosis.